A test method using a DNA sequencer or the like is practically used for the diagnosis of genetic diseases caused by genetic malformation or diseases caused by DNA mutation or the like such as various types of cancer. On the other hand, the decoding of the genomic base sequences of various types of organisms has progressed, and based on the findings, research and development aiming at analyzing gene function have further vigorously been progressing.
Incidentally, for analysis of gene function, there is required the development of techniques of efficiently measuring and testing individual difference or mutation of a genetic sequence, the frequency of gene expression in a cell, etc. Among such techniques, as one of methods for testing cancer or the like, there is known a method for examining the expression of a specific gene, which comprises adding a test sample consisting of a DNA fragment extracted from a subject and labeled with fluorescence or radioisotope onto a DNA microarray on which a given target DNA fragment is mounted in advance, performing hybridization, identifying and recognizing fluorescent- or radioisotope-labeling on a specific spot, so as to examine whether or not the specific gene expresses in the test sample.
In a step of forming a complementary double strand from a DNA fragment immobilized on a microarray and a DNA probe labeled with fluorescence or radioisotope (hereinafter, referred to as an “annealing step”), various conditions such as temperature, pH and salt concentration are set as appropriate depending on the base composition of used DNA. For example, a temperature condition is set using the melting temperature (hereinafter, referred to as “Tm”) of DNA as an indicator.
Generally, where DNA has a fixed length, as the GC content represented by the total of the content rates of guanine and cytosine that are the types of bases constituting DNA increases, the Tm value increases. Moreover, the temperature of annealing differs depending on the length of the repeat sequence of DNA and the structural complexity of repeat number. Accordingly, the optimal temperature of annealing is specific for a DNA fragment, and the optimal temperature of annealing differs depending on a DNA probe used.
Furthermore, factors of determining conditions for annealing include pH, salt concentration or the like other than the above described temperature, but in any cases, the optimal conditions differ depending on the type of a DNA fragment used. That is to say, when multiple kinds of DNA probes are used, it is difficult to undifferentiatedly determine the conditions for annealing. Accordingly, in many cases, it is common that hybridization is carried out under conditions such as a low temperature, neutral pH and a high salt concentration, where annealing can be performed relatively easily, and then gene expression is analyzed.
However, since even DNAs having low complementarity are annealed with each other under the above described annealing conditions, there is a need to eliminate double stranded DNA having low complementarity in a washing operation following the annealing. Therefore, there may occur problems that a washing performed under stringent conditions results in poor detection sensitivity or the like.
In contrast, if a washing operation is carried out moderately to increase detection sensitivity, the noise signal from background increases, the number of pseudo-positive increases, and thereby it becomes difficult to perform an accurate test and analysis. So, detection sensitivity may be lowered depending on used DNA probes and test samples, and therefore the development of a hybridization method having higher detection sensitivity has been desired for highly sensitive gene expression analysis.
The present invention has been made to solve such problems of the prior art, and it is an object of the present invention to provide a method for hybridizing nucleic acids, which has high measurement sensitivity with simple operation, and a method for gene analysis using the above method for hybridizing nucleic acids.